2.1. Heparin and Heparin Binding Growth Factors
Heparin, a mucopolysaccharide, is a constituent of various tissues, especially liver and lung, and mast cells in several mammalian species. Chemically, it has been described as an .alpha.,.beta. glycosidically linked sulfated copolymer of D-glucosamine and D-glucuronic acid. It is well known as an anticoagulant that functions by accelerating the rate at which antithrombin III inactivates the proteases of homeostasis (Jacques, 1980, Pharmacol. Rev. 31: 99-166).
The exact structure of heparin and the precise nature by which it acts in blood anticoagulation however has not been discovered. Much of the difficulty in determining the structure of heparin results from its complexity and the fact that it is not a homogeneous, well-defined substance (Barzu et al., 1985, Biochem. Biophys. Acta 845: 196-203; Linhardt et al., 1982, J. Biol. Chem. 257: 7310-7313; Lindahl et al., 1977, Fed. Proc. 36: 19-23; and Laurent et al., 1978, Biochem. J. 175: 691-701). Heparin is also polydisperse with a molecular weight range from about 5,000 to 40,000. Within a given chain, there are also structural variations such as the varying degrees of sulfation, N-acetylation and C-5 epimerization in the uronic acid residue.
Heparin has also been found to coact with cortisone to inhibit angiogenesis (described in U.S. patent application Ser. No. 641,305 filed Aug. 16, 1984, the contents of which are incorporated herein by reference). Angiogenesis is the induction of growth of new capillary blood vessels and is important in normal processes such as development of the embryo, formation of the corpus luteum, and wound healing. It is also an important component in pathological processes such as chronic inflammation, certain immune responses, and neoplasia. Administration of heparin and cortisone to mice with certain kinds of tumors inhibits the generation of essential capillary vessels that support tumor growth, and can cause the collapse of the blood supply which supports the tumors (see Folkman, 1986, Cancer 46: 467 for a review of the history of this discovery and related subject matter).
A major disadvantage in the use of heparin with a steroid to inhibit angiogenesis results from the fact that heparins manufactured by different processes and different companies revealed quite different antiangiogenic activities despite similar anticoagulant activities. The precise composition of commercial heparin apparently varies depending on its source and method of manufacture. This variation could create problems when heparin is used for other purposes.
A number of endothelial cell growth factors, now known as heparin binding growth factors (HBGFs) have been found to have a strong affinity for heparin (reviewed in Lobb, 1988, Eur. J. Clin. Invest. 18: 321-328 and Folkman and Klagsbrun, 1987, Science 235: 442-447). As will be discussed in further detail infra, the purification of HBGFs was greatly facilitated by heparin affinity chromatography. Recently, it has been shown that heparin and the heparin like molecule, hexuronyl hexosaminoglycan sulfate (HHS-4) can protect basic or acidic fibroblast growth factor from acid or heat activation (Gospodarowicz and Cheng, 1986, J. Cell. Phys. 128: 475-484). Heparin has also been found to protect ECGF from proteolytic inactivation.